Hsp90 regulates EphA2 signaling and cell migration in glioblastoma. The majority of brain cancers manifest as high-grade glioblastomas (GBMs), which are ultimately fatal within 15 months from time of diagnosis. GBM cells are extremely invasive and distal infiltration into normal brain tissue leads to the formation of secondary aggressive tumors and death. Newer treatments that focus on the molecular underpinnings of GBM are urgently needed. Cell migration is central to the processes of cell invasion and angiogenesis, both of which are integral to GBM progression. The receptor tyrosine kinase EphA2 is highly overexpressed in aggressive solid tumors such as GBM, and it plays a major role in facilitating cell migration. EphA2 coordinates signaling from integrin receptors and modulates cell motility in a pathway involving FAK and downstream intermediates, culminating in cytoskeletal rearrangements required for cell motility. Therefore, the goal of this proposal is to target EphA2 in GBM as an approach to curtail cell migration and to impact upon the invasiveness of this disease. Hsp90 is a molecular chaperone required for the folding and activation of numerous substrates, of which kinases comprise a disproportionate percentage. Pharmacologic inhibition of Hsp90 with geldanamycin (GA) leads to the destabilization and depletion of Hsp90 substrate proteins. Hsp90 inhibitors possess anti-tumorigenic properties and diminish cell migration and cell invasion, although the targets involved are not well defined. We demonstrate that Hsp90 is required for EphA2 activity and that GA depletes receptor levels in GBM cell lines and reduces cell motility. Thus, we highlight a novel pharmacologic opportunity to target EphA2 protein. Depletion of EphA2 via ligand addition elicited similar effects in curtailing cell migration, implicating EphA2 as a major effector for Hsp90's role in GBM cell migration. We will further delineate the role of cellular Hsp90 in mediating EphA2 signaling (Aim 1), which will provide information potentially relevant to future clinical trial design. We also report that inhibition of cell surface localized Hsp90 potently inhibits GBM motility and that these effects are at least partially mediated through EphA2. We will determine the mechanistic basis for cell surface-dependent regulation of EphA2 and cell motility (Aim 2). It is our goal to obtain a more complete understanding of the distinct functions of intracellular and surface-localized Hsp90 relevant to their roles in EphA2 signaling and GBM cell migration. This understanding is critical for the design of future therapeutics, as it may be optimal to target the surface- localized chaperone, thus sparing its intracellular functions. Finally, we will test whether targeting EphA2 via ligand or Hsp90-based approaches have efficacy in an in vivo orthotopic intracranial model of GBM. These studies take us from an initial discovery of Hsp90-dependent regulation of EphA2 to a relevant preclinical animal model. Collectively, these studies will shed light on critical pathways involved in GBM cell migration and will potentially lead to rational molecular-targeted therapeutic approaches to diminish the invasive nature of this disease and to improve clinical outcome. PUBLIC HEALTH RELEVANCE: High-grade glioblastoma (GBM), represents the most common form of brain cancer and is ultimately fatal within 15 months from time of diagnosis. Therefore, the goal of this proposal is to target one of the key oncogenic proteins involved in GBM progression as an approach to curtail cell migration and to impact upon the invasiveness of this disease. Our studies will potentially lead to rational molecular-targeted therapeutic approaches to improve GBM survival.